【作者】 刘国兰；

【导师】 罗利军； 梅捍卫；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 干旱是世界农业生产的主要障碍之一,水稻是世界上主要耗水作物,水稻抗旱性的研究对粮食增产增收具有重要意义。建立科学有效的抗旱性评定方法和鉴定体系,定位与克隆水稻抗旱性相关的基因,利用分子标记辅助选择和转基因技术,结合常规育种,进行节水抗旱、优质高产多抗等性状的聚合,培育节水抗旱新品种是水稻抗旱性研究的主要内容。本研究对可能的抗旱鉴定指标穗颈粗和穗水势进行了分析,利用抗旱群体材料对抗旱性状进行分子标记定位,并通过标记辅助选择技术构建了4个抗旱目标区段的近等基因系,精细定位了部分相关主效OTL,通过候选基因法克隆了其中一个可能的抗旱相关基因QsGRAS,并将来源于第4染色体的目标区段导入到早稻保持系中,获得了稳定株系。主要结果如下:1.穗水势:调查生育后期进行干旱处理的3个品种(2个旱稻和1个水稻)在晴天每隔1-1.5h一天内穗水势和叶水势的变化,发现旱稻品种的穗水势和叶水势比水稻品种开始下降要慢,比水稻品种保持更高的水平且恢复更快。在晴天中午,穗水势和叶水势在旱稻和水稻间的差异甚至在正常水分条件下也能观察到。穗水势和叶水势与关乎植株生长和植株产量组成因子的7个性状存在相似的相关性。基于穗水势和叶水势日变化的的平行趋势及两者间的高度相关,提出穗水势可以作为植株水分状况的一个指标,穗水势能更有效区分不同旱稻品种的抗旱性差异。2.穗颈粗及种质创新:调查以珍汕97B和IRAT109构建重组自交系群体在不同水分处理下的穗颈粗,发现穗颈粗与很多农艺性状,尤其是穗型极显著相关。通径分析表明两种水分条件下穗颈粗主要是通过颖花数或实粒数来对产量产生影响。基于213个SSR标记构建的连锁图谱,总共检测到3个控制穗颈粗的主效QTL,其中qPND-4在两种水分条件下都检测到,贡献率为5.03%.11.01%。在第4染色体RM241-RM349标记区间,共定位到7个穗型相关的QTL,控制穗数、穗长、一次枝梗数、二次枝梗数、每穗颖花数、着粒密度和每穗实粒数。共检测到17个与穗颈粗有关的上位性QTL,贡献率变异为2.58%-5.64%。通过杂交和连续回交,利用分子标记辅助选择技术对前景进行筛选,将IRAT109的第4染色体区段导入到旱稻不育系沪旱1A的保持系沪旱1B中,抗旱性鉴定发现,导入的材料在实粒数、穗颈粗和产量上有显著提高。3.近等基因系构建与QsGRAS基因克隆:立足于两年定位结果中5个控制较多抗旱相关性状的QTL区段,通过杂交和回交,并利用分子标记对前景和背景进行选择,构建目标区段来源于早稻IRAT109而其余背景均为水稻珍汕97B的近等基因系。对其中4个区段的近等基因系进行了抗旱性鉴定,在干旱条件下导入有OTL区段的近等基因系在产量和结实率上均高于珍汕97B。对第4染色体的RM273-RM255和第7染色体的RM134-RM420通过加密SSR和ILP标记,将控制实粒数和产量的OTL缩小到95kb,穗数和颖花数的QTL定位在609kb的区间内,结实率定位在227kb的范围内。第7染色体上影响实粒数和结实率的QTL定位在83kb的区间内,而控制颖花数和实粒数的则定位在446kb范围内。对精细定位的区段内的候选基因进行了初步的分析。并根据候选基因cDNA序列,设计引物,扩增获得OsGRAS的编码cDNA和启动子序列。对其保守区域进行分析,发现OsGRAS是GRAS转录因子家族的一个成员,其具有该家族典型的结构域。通过酶切及载体连接,共构建了OsGRAS基因及其启动子相关的三个表达载体。更多还原

【Abstract】 Drought is one of the most serious world-wide problems in agriculture, rice is the largest water consumer in crops. Development of drought tolerant varieties is largely based on the quick and precise screening of germplasm and breeding materials in water-limited environments, mapping and cloning the drought-tolerance gene, incorporating the gene of high-yield, good-quality and drought tolerance by using MAS and transgenic technology. In this study, we analysed the characteristics of drought tolerance including panicle neck diameter and panicle water potential; mapped QTLs affecting drought tolerance in a RIL population; developed four sets of QTL-NILs by MAS and evaluated their DT ability; cloned a DT gene named OsGRAS by candidate-gene strategy; and applied QTL mapping result to the breeding program by introgressing the target region from chromosome 4 into upland rice CMS maintainer line "Huhan1B". Main results of the present study are as following:1. Study on panicle water potential. Two upland varieties (IRAT109, IAPAR9) and one lowland variety (Zhenshan 97B) were grown in summer season and treated by drought stress and normal condition in reproductive stage. Panicle water potential (PWP) and leaf water potential (LWP) were measured every l-1.5h in 24h in sunny days. Both PWP and LWP of upland varieties started to decreased later in the morning, maintained in a higher level at solar noon and recover more quickly in the evening than that of lowland variety. The results showed that PWP can be used as an indicator of plant water status based on the parallel daily changes, and the high correlation between PWP and LWP. Similar correlations were also observed between PWP, LWP and seven traits about plant growth and grain yield formation. PWP seemed to be more effective to distinguish the upland rice varieties with different drought tolerant ability. Difference of PWP and LWP between upland and lowland rice varieties were also observed in solar noon even under normal water condition.2. Study on panicle neck diameter and DT target interval introgressed into "Huhan1B" by MAS. The panicle neck diameter was measured in a rice RIL population of 187 lines from the cross "Zhenshan97B/IRAT109" under different water conditions in 2003 and 2004. Panicle neck diameter was found to be significantly correlated with many agronomical traits, especially with the panicle size. It was found by path analysis that panicle neck diameter has effect on yield by increasing the spikelet number and grain number. Based on a linkage map of 213 SSR markers, three main effect QTLs were detected for PND with contribution rates varied from 5.03% to 11.01%, including one locus detected under both water conditions (qrPND-4). The chromosomal region on chromosome 4 (RM241-RM349) also hosted QTLs for seven panicle traits, including panicle number, panicle length, primary branch number, secondary branch number, spikelet number per panicle, spikelet density and grain number per panicle. Seventeen epistatic QTLs were detected for PND under well-watered and stress conditions. The contribution rate of each pair of epistatic effect varied from 2.58% to 5.64%.During three times backcross procedure, positive individuals were selected in the progeny by foreground markers for further crossing or selfing. The target interval of chromosome 4 from IRAT109 was introgressed into an upland maintainer line "Huhan1B". It was found that introgressed lines have more grain number, higher yield and thicker panicle neck diameter but less panicle number than "Huhan1B" under drought stress.3. NIL Development and gene cloning of OsGRAS by candidate-gene strategy. Five QTLs controlling several DT traits was selected according to the mapping results for two years. NILs was developed by crossing and continuous backcross, and progeny selection by both foreground and background markers. The drought tolerance ability was evaluated for four populations of QTL-NILs. The NILs has higher yield and spikelet fertility than Zhenshan97B as the recurrent parent. Fine mapping was done to narrow down the interval by adding more SSR and ILP markers for the interval RM273-RM255 on chromosome 4 and RM134-RM420 on chromosome 7. For the target interval of chromosome 4, QTLs of both grain number and grain yield located in the interval RM17355-RM17352 (95kb), QTL of panicle number and spikelet number located in the interval RM5320-RM17355 (609kb), QTL of spikelet fertility located in the interval RM17183-RM17190 (227kb). For the region RM134-RM420 of chromosome 7, we found that QTLs influencing both grain number and spikelet fertility were located in RM134-RI04226 (83kb), spikelet number and grain yield located within RM1306-RI04046 (446 kb).Based on the sequence of candidate gene, full-length cDNA and promotor sequnece was obtained by primer design and PCR amplification. OsGRAS is a member of the GRAS Family of transcriptional regulators. OsGRAS has the typical domain of GRAS family accoding to the conserved domain alignment.Three expression vectors of OsGRAS were constructed by enzyme digestion and vector ligation.更多还原